Project Summary Opioid analgesics are the mainstay of treatment for severe pain caused by cancer and by tissue and nerve injury. However, clinical use of ?-opioid receptor agonists can cause hyperalgesia and the loss of analgesic efficacy, which lead to opioid dose escalation and remain the major obstacles to adequate pain relief with opioids. Although some evidence suggests a strong link between hyperalgesia and analgesic tolerance induced by opioids, the unifying cellular and molecular mechanisms for these two important phenomena are poorly understood. The major objective in this proposal is to identify the essential signaling mechanisms responsible for opioid-induced hyperalgesia and tolerance (OHT). Opioid administration can cause a persistent increase in glutamate release from TRPV1-expressing primary afferents through stimulation of presynaptic N-methyl-D-aspartate receptors (NMDARs), which represents a key mechanism for OHT. NMDARs are a clinically validated target for treating OHT, but little is known about how opioids lead to increased presynaptic NMDAR activity at the spinal cord level. Also, gabapentinoids can reduce OHT in animal models and in patients through a largely unknown mechanism. Our preliminary data showed that phospholipase C was critically involved in increased spinal NMDAR activity and in OHT. Furthermore, opioid-induced phospholipase C stimulation promoted the interaction between ?2?-1 and NMDARs, and such an interaction was diminished by gabapentin. In this project, we will test our central hypothesis that phospholipase C-??dependent signaling contributes to the development of OHT predominantly by promoting ?2?-1?NMDAR interaction to increase presynaptic NMDAR activity at the spinal cord level and that gabapentin reduces OHT and spinal presynaptic NMDAR activity by interrupting ?2?-1?NMDAR interaction. We will use a multidisciplinary approach, including protein biochemistry, electrophysiological recordings in spinal cord slices, siRNA knockdown, and genetic ?2?-1 and phospholipase C-? isoform knockout mice. The proposed studies are highly significant because it will greatly advance our understanding of the fundamental signaling mechanism involved in OHT. Our findings could also help developing new therapeutic agents targeting PLC-? isozymes and ?2?-1?NMDAR interaction sites for treating OHT and reducing opioid consumption and dependence in patients.